The experiments carried out in this Section have led to an idea that the covalent structures of native proteins carry the information for specific global cooperative interactions operational only in uniquely folded structures to stabilize the structures. These unique structure would be the native three-dimensional structures. It would follow that if the three-dimensional structure is evolutionarily conserved, the covalent structure would have evolved in conserving information for the global cooperative interactions. Cytochrome c contains two unique covalent structures, the amino acid sequence and two thioether bonds between a heme moiety and apocytochrome c. With respect to these two thioether bonds, 8 stereochemical isomers are possible. We have found and solubilized an enzyme, cytochrome c synthetase from yeast mitochondria and established that the enzyme catalyzes direct attachment of heme to apocytochrome c through two thioether bonds to form cytochrome c. To find whether only one of 8 possible isomers being conserved by this enzymatic action, we continue purification and characterization of the enzyme. Only limited regions of the amino acid sequences are permissible for cleavage without disruption of the hypothetical global cooperative interactions. Such permissible regions of horse cytochrome c are found to be between residues 23 and 25, residues 37 and 38, and residues 53 and 54. The results also indicate a possibility that these permissible regions may be universal in eucaryotic cytochrome c and therefore conserved during evolution. As reported in the previous year, cytochrome c synthetase of yeast mitochondria catalyze attachment of heme to apofragment of residues 1 to 25 of horse cytochrome c. Thus, using cytochrome c synthetase, chemical synthesis of cytochrome c with and without substituted residues will be carried out to investigate conservation of the global cooperative interactions on the basis of the permissible regions. The goal of this project is to obtain an insight into evolution and conservation of mechanism of global cooperative interactions underlying the three-dimensional structures of proteins to give the function.